The aim of the project is to elucidate mechanisms behind the development of tolerance and dependence to opioids. Opioids interact with specific receptors in the CNS and in the gut. Binding assays show at least two different populations of receptor sites. One with selectivity for narcotic agonists, the other for antagonists. We have found an endogenous substrate for the agonist sites which is a small peptide, MLF, which can be extracted from brain of various animal species and from human CSF. In the morphine tolerant animal we have evidence for the formation of another peptide. NLF, with selectivity for antagonist sites. It seems highly probable that these factors are involved in the development of morphine tolerance and dependence. I now intend to measure the rate of synthesis and the absolute levels of these factors in relation to morphine tolerance and dependence in rats and dogs. At present, assays are made in radio-receptor systems but efforts will be made at large-scale preparation of the factors and the development of radioimmunoassays. The rate of synthesis and its localization will be studied by the use of isotope labeling with precursor amino acids. When purified material is available it will be tested for biologic effects after local administration. Radiolabeled peptides will be tested in receptor systems to define the characteristics of their interaction with opioid receptors and compare those with those of morphine congeners. I will approach the physiological and clinical significance of these peptides by measuring the levels in CSF from patients with various diagnoses, such as severe pain, psychiatric and neurological diseases. This study will be extended to human addicts and, if possible, to patients undergoing electroanalgesic treatment.